摘要
目的 目前细菌定量检测提取细菌基因组多采用裂解法,此方法在提取基因组过程中部分基因组丢失。荧光定量PCR定量检测细菌常采用纯化的PCR产物制备标准品,但用这种标准品制备标准曲线没有考虑基因组提取过程中基因组丢失问题,检测结果偏低。本研究使用细菌直接计数法制备标准品,力争减少提取基因组过程中部分基因组丢失造成的误差。方法脆弱类杆菌作为试验菌株,用细菌直接计数法和纯化PCR产物制备标准品,检测用细菌计数法获得的细菌基因组,用统计学软件分析两种方法得到的检测结果。结果用两种不同的标准品作荧光定量PCR检测1000个细菌基因组μL-1的标本,细菌计数法制作标准品测得的结果是763.8个细菌基因组μL-1,普通PCR产物制作标准品测得的结果是112.9个细菌基因组μL-1,统计软件SPSS10.0分析结果表明,使用两种不同的标准品,荧光定量PCR检测结果差别显著(p<0.05)。结论使用细菌直接计数法制备标准品比用PCR产物制备标准品作荧光定量PCR检测细菌标本的结果更接近实际结果。
Objective At present, the cracking method is often used when genome is extracted in quantification detection of bacteria. The part genome DNA can be losed when we extract genome DNA with this method. In detection bacteria with FQ - PCR assay, purified product of PCR is used as standard sample, however, the problem of genome lose is ignored. The objective of study is to use the other method （ directly counting bacteria method ） to make standard sample to reduce error that be caused by genome lose. Method The standard sample was made by the method of directly counting bacteria of B. fragilis. Respectively we detected the same sample using standard sample obtained by method of directly count bacteria and standard sample obtained through product of conventional PCR, then we analyzed the results obtained by the above two different methods by statistics software （ SPSS10.0）. Result The 764.9 genome μL^-1 was obtained through detecting the sample containing 1000 bacteria genome μL^-l by FQ -PCR assay using directly counting bacteria as standard sample, the 112.9 genome μL^-1 was the result using product of PCR as standard sample. The first number of genome was higher than the latter number signifieantly （ p 〈 0. 05 ）. Conclusion The result of FQ - PCR using standard sample obtained by method of directly counting bacteria was closer to the real result than The result using product of conventional PCR as standard sample.
出处
《河南预防医学杂志》
2007年第1期1-3,共3页
Henan Journal of Preventive Medicine
基金
河南科技攻关项目(0523031700)
